ABSTRACT Hepatocellular carcinoma (HCC) is among the top four leading causes of cancer-related death worldwide with nearly 700,000 deaths per year, and has increased in recent years in developed nations. Long noncoding RNA (lncRNA) may regulate nuclear gene/protein function of liver tumor-initiating stem-like cells (TICs), which have self-renewal properties and are refractory to chemotherapy, leading to relapse and metastasis. MSI2 and MYC are upregulated in 70% of HCC patients. MYC is a pioneer pluripotent transcription factor that reprograms gene expression in differentiated cells. From preliminary studies: (1) Our MSI2 RIP-seq analysis established that MSI2 binds to c-MYC mRNA and long non-coding RNA (lncRNAs), including microRNA-22 host gene (miR-22hg) and MALAT1. (2) MSI2 inhibited processing of miR22hg and enhanced c-MYC translation via binding to its 5'-IRES (Internal Ribosome Entry Site). (3) MSI2 induction in liver promoted HCC development in inducible-MSI2 mice (ROSA26rtTA; Alb-TetO-Msi2 mice). Our hypothesis is that MSI2-binds to lncRNAs and c-MYC primary mRNA transcripts to change 3D chromatin structure and long-range chromatin interactions resulting in the genesis of TICs through c-MYC/super-enhancer-mediated reprogramming with lncRNAs. Thus, the goal of this proposal is to understand better how the MSI2 pathway and super-enhancer cooperatively mediate the genesis of TICs in alcohol-associated HCC patients. Targeting of the TIC subpopulation and super-enhancer function should effectively eliminate the TIC-derived HCCs. To test this hypothesis, the following two aims are proposed; In Aim 1, we will test if MSI2 promotes stability and increased translation of c-MYC mRNA by binding to lncRNAs, including miR22hg and MALAT1. We will test in a cell culture model whether MSI2-binding to c-MYC mRNA and lncRNA (including miR-22hg/pri-miR22, MALAT1 and enhancer RNA) stabilizes c-MYC mRNA resulting in increased translation. In a subaim, we will delineate the super-enhancer regions and transcriptionally active genomic loci containing MSI2 and NANOG by integrated analyses of Global Run-On sequence (GRO-seq) assay with Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) assay under collaborative effort with Dr. Rosenfeld (HHMI, UCSD). In Aim 2, we will test the therapeutic effects of selective TIC inhibitor plus super-enhancer inhibitor to counter the increased expression of c-MYC resulting from the super-enhancer complex in alcohol-associated HCC mouse models: In vivo experiments will be performed by treatment with TIC inhibitor and/or super-enhancer-inhibitor (BET-bromodomain inhibitor JQ1) in alcohol-fed, FRG-hu-hep-HSC humanized mouse models. We will determine if lncRNAs found in primary liver tumors regulate tumor formation at distant metastatic sites. The proposed research will have a broader impact by identifying the mechanism by which MSI2 inhibits miR22hg processing and will validate how alcohol induces MSI2 for liver cancer.